Hui Su Lee

Comparison of the Biological Activity Between Ultrafine and Fine Titanium Dioxide Particles in RAW 264.7 Cells Associated with Oxidative Stress

Ultrafine or fine titanium dioxide (TiO2) particles are widely used in the production of white pigments, for sunscreens, and in cleanup techniques. However, currently knowledge is deficient concerning cellular responses to these particles. The study evaluated and compared the biological activity of ultrafine and fine TiO2 particles in RAW 264.7 macrophages according to an oxidative stress paradigm. In vitro exposure of macrophages to ultrafine or fine TiO2 in the range of 0.5–200 μg/ml did not significantly alter cell viability. However, ultrafine TiO2 enhanced intracellular generation of reactive oxygen species (ROS) to a greater extent than fine TiO2 at each exposure concentration. Ultrafine TiO2 induced ERK1/2 activation in a concentration-dependent manner, while the fine TiO2-induced changes were minimal. Phosphorylation of ERK1/2 occurred following 10 min exposure to higher concentrations of ultrafine TiO2 (≥25 μg/ml). Similarly, ultrafine TiO2 exposure significantly enhanced tumor necrosis factor (TNF)-α and macrophage inflammatory protein (MIP)-2 secretion in a concentration-dependent manner, and its potency was higher than fine TiO2. These findings suggest that when exposure concentration is based upon equivalent mass, ultrafine TiO2 exerts greater biological activity as measured by ROS generation, ERK 1/2 activation, and proinflammatory mediator secretion in RAW 264.7 macrophages than fine TiO2.

Inhibition of c-Jun NH2-terminal kinase or extracellular signal-regulated kinase improves lung injury

BackgroundAlthough in vitro studies have determined that the activation of mitogen-activated protein (MAP) kinases is crucial to the activation of transcription factors and regulation of the production of proinflammatory mediators, the roles of c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) in acute lung injury have not been elucidated.MethodsSaline or lipopolysaccharide (LPS, 6 mg/kg of body weight) was administered intratracheally with a 1-hour pretreatment with SP600125 (a JNK inhibitor; 30 mg/kg, IO), or PD98059 (an MEK/ERK inhibitor; 30 mg/kg, IO). Rats were sacrificed 4 hours after LPS treatment.ResultsSP600125 or PD98059 inhibited LPS-induced phosphorylation of JNK and ERK, total protein and LDH activity in BAL fluid, and neutrophil influx into the lungs. In addition, these MAP kinase inhibitors substantially reduced LPS-induced production of inflammatory mediators, such as CINC, MMP-9, and nitric oxide. Inhibition of JNK correlated with suppression of NF-κB activation through downregulation of phosphorylation and degradation of IκB-α, while ERK inhibition only slightly influenced the NF-κB pathway.ConclusionJNK and ERK play pivotal roles in LPS-induced acute lung injury. Therefore, inhibition of JNK or ERK activity has potential as an effective therapeutic strategy in interventions of inflammatory cascade-associated lung injury.

Inhibition of SRC Tyrosine Kinases Suppresses Activation of Nuclear Factor-κB, and Serine and Tyrosine Phosphorylation of IκB-α in Lipopolysaccharide-Stimulated Raw 264.7 Macrophages

Involvement of protein tyrosine kinases (PTK) in lipopolysaccharide (LPS)-induced nuclear factor-kappa B (NF-κB) activation has been demonstrated. Studies investigated the role of PTK and the underlying mechanisms by which PTK play a role in LPS induction of pathways leading to NF-κB activation in macrophages. Inhibitors of PTK—genistein, herbimycin A, or AG126—blocked LPS-induced NF-κB activation. Genistein also blocked pervanadate-induced NF-κB activation. Furthermore, Src TK selective inhibitors—damnacanthal or PP1—blocked LPS-induced NF-κB activation over a range of nanomolar concentrations. Genistein, damnacanthal, or PP1 blocked the LPS-induced serine phosphorylation, the degradation of IκB-α, and the consequent translocation of the p65 subunit of NF-κB to the nucleus. In addition to serine phosphorylation of IκB-α, LPS-induced NF-κB activation also required tyrosine phosphorylation of IκB-α. These TK inhibitors blocked substantially LPS induction of tyrosine phosphorylation of IκB-α. Furthermore, cSrc and Lck were physically associated with IκB-α. These results suggest that the LPS-induced NF-κB pathways are dependent on both serine and tyrosine phosphorylation of IκB-α, and that Src TK, such as cSrc and Lck, are key components of the LPS signaling pathway through at least two different mechanisms associated with NF-κB activation. This work was supported by grant R04-2002-000-00023-0 from the Basic Research Program of the Korea Science and Engineering Foundation.

Time course for inhibition of lipopolysaccharide-induced lung injury by genistein: Relationship to alteration in nuclear factor-κB activity and inflammatory agents*

ObjectiveThis study determined the time course for inhibition of lipopolysaccharide-induced acute lung injury following a single dose of genistein. In addition, the study investigated whether a multiple dosing schedule with genistein retained the inhibitory effects on acute lung injury, nuclear factor-&kgr;B activation, and production of nuclear factor-&kgr;B-dependent inflammatory agents, such as matrix metalloproteinase-9 and nitric oxide. DesignProspective, randomized, laboratory study. SettingExperimental laboratory at a university. SubjectsRats weighing 280–300 g. InterventionsSaline or lipopolysaccharide (6 mg/kg of body weight) administered intratracheally with a single dose of genistein (50 mg/kg) or a multiple dosing schedule with genistein (16 mg/kg every 6 hrs for 2 days with lipopolysaccharide treatment at 24 hrs after the first administration of genistein). Measurements and Main ResultsA 2-hr pretreatment with genistein (a single dose) inhibited biochemical lung injury variables as well as neutrophil infiltration with a maximal inhibition at 4 hrs after lipopolysaccharide treatment. These inhibitory effects of genistein declined with time and were no longer significant by 14–24 hrs after lipopolysaccharide treatment. The multiple dosing schedule with genistein retained significant inhibitory effects on biochemical lung injury variables and the number of neutrophils in the bronchoalveolar lavage fluid at 24 hrs after lipopolysaccharide treatment compared with a single pretreatment with genistein. The multiple dosing schedule with genistein also enhanced the inhibition of induced nuclear factor-&kgr;B activity as well as matrix metalloproteinase-9 activity and nitric oxide production at 24 hrs after lipopolysaccharide treatment. ConclusionsThis study reports the time course of the inhibitory effects of a single genistein pretreatment on acute lung injury with the maximal effects at 4 hrs after lipopolysaccharide treatment. However, a multiple dosing schedule with genistein retained the inhibitory effect on acute lung injury at 24 hrs after lipopolysaccharide treatment. The mechanisms by which genistein exerts an inhibitory effect on acute lung injury may involve the suppression of nuclear factor-&kgr;B activation, matrix metalloproteinase-9 activity, and NO production.

Inhaled nitric oxide down-regulates intrapulmonary nitric oxide production in lipopolysaccharide-induced acute lung injury.

Objective To examine whether inhaled nitric oxide (NO) affected the intrapulmonary production of NO, reactive oxygen species, and nuclear factor-&kgr;B in a lipopolysaccharide (LPS)-induced model of acute lung injury. Design Prospective, randomized, laboratory study. Setting Experimental laboratory at a biomedical institute. Subjects Twenty male rabbits weighing 2.5–3.5 kg. Interventions Saline or LPS (5 mg/kg of body weight) was administered intravenously with or without NO inhalation (10 ppm) in each group of five rabbits. Measurements and Main Results LPS increased the lung leak index, the neutrophils and NO levels in bronchoalveolar lavage fluid, and NO levels produced by resting and stimulated alveolar macrophages. Inhaled NO decreased the lung leak index, the neutrophils and NO levels as measured by nitrite levels in the lavage fluid, and NO produced by the resting and stimulated alveolar macrophages. Inhaled NO also blocked the activities of reactive oxygen species and nuclear factor-&kgr;B binding to DNA in lavage cells and in alveolar macrophages. Conclusion Inhaled NO attenuates LPS-induced acute lung injury, possibly by decreasing NO production in the lungs. The mechanism of reducing NO production resulting from inhaled NO may involve, in part, the activities of reactive oxygen species and/or nuclear factor-&kgr;B.

Phosphoinositide 3-kinase activity leads to silica-induced NF-κB activation through interacting with tyrosine-phosphorylated IκB-α and contributing to tyrosine phosphorylation of p65 NF-κB

The role of the subunits of phosphoinositide (PI) 3-kinase in NF-κB activation in silica-stimulated RAW 264.7 cells was investigated. Results indicate that PI3-kinase activity was increased in response to silica. The p85α subunit of PI3-kinase interacted with tyrosine-phosphorylated IκB-α in silica-stimulated cells. PI3-kinase specific inhibitors, such as wortmannin and LY294003, substantially blocked both silica-induced PI3-kinase and NF-κB activation. The inhibition of NF-κB activation by PI3-kinase inhibitors was also observed in pervanadate-stimulated but not in LPS-stimulated cells. Furthermore, tyrosine phosphorylation of NF-κB p65 was enhanced in cells stimulated with silica, pervanadate or LPS, and wortmannin substantially inhibited the phosphorylation event induced by the first two stimulants but not LPS. Antioxidants, such as superoxide dismutase (SOD), N-acetylcysteine (NAC) and pyrrolidine dithiocarbamate (PDTC), blocked silica-induced PI3-kinase activation, suggesting that reactive oxygen species may be important regulatory molecules in NF-κB activation by mediating PI3-kinase activation. Our data suggest that p85 and p110 subunits of PI3-kinase play a role in NF-κB activation through interaction with tyrosine-phosphorylated IκB-α and contributing to tyrosine phosphorylation of p65 NF-κB.

IRON TETRAKIS (N-METHYL-4'-PYRIDYL) PORPHYRINATO (FeTMPyP) IS A POTENT SCAVENGING ANTIOXIDANT AND AN INHIBITOR OF STIMULANT-INDUCED NF-?B ACTIVATION OF RAW 264.7 MACROPHAGES

Metalloporphyrins have been shown to be protective in oxidative stress models. However, the molecular basis for the antioxidative and antiinflammatory activities of iron tetrakis (N

Inhibition of Mitogenic Stimulant-Induced Activation of Thymocytes with Zinc Tetrakis-(N-Methyl-4′-Pyridyl) Porphyrinato

Zinc porphyrins have anti-inflammatory and anti-allergic properties. The objective of the present study was to characterize the mechanism of zinc tetrakis-(N-methyl-4′-pyridyl) porphyrinato (ZnTMPyP) immune modulation by investigating its effects on the proliferative activity during thymocyte stimulation with mitogenic factors and the molecular events mediating thymocyte proliferation. The results indicate that ZnTMPyP inhibited thymocyte proliferation stimulated with various mitogenic factors, such as concanavalin A (Con A), interleukin (IL)-1β, and lipopolysaccharide-exposed macrophage supernatant, in a concentration-dependent manner. ZnTMPyP was also effective in preventing DNA binding activity of nuclear factor κB (NF-κB) and IL-2 production by thymocytes in response to Con A or IL-1β. Inhibition of p38 mitogen-activated protein kinase (MAPK) with SB203580 substantially inhibited Con A- or IL-1β-induced DNA binding activity of NF-κB, whereas ZnTMPyP inhibited the activation of p38 MAPK. ZnTMPyP also inhibited Con A-induced chemiluminescence and tyrosine phosphorylation by thymocytes. In conclusion, our findings suggest that the antiproliferative effect of ZnTMPyP may be mediated by effective inhibition of the production of reactive oxygen species, tyrosine phosphorylation, p38 MAPK activation, NF-κB activation, and IL-2 production during mitogenic stimulation of thymocytes.